Deletion and glycosylation mutant of human tissue plasminogen activator

ABSTRACT

Thrombolytic proteins are disclosed which have tissue plasminogen-type activity. The proteins are characterized by modification within the 94 amino acid N-terminus, and/or at Arg-275, and/or at one or more of the N-linked glycosylation sites. Methods for making these proteins are disclosed as are therapeutic compositions containing same.

This application is a continuation of Ser. No. 07/382,678, filed Oct.19, 1988, presently issued as U.S. Pat. No. 5,071,972. Application Ser.No. 07/382,678 was based upon PCT application Ser. No. PCT/US87/00267,filed Jan. 30, 1987 and was a continuation-in-part of the following U.S.patent applications: Ser. No. 861,699, filed May 9, 1986, presentlyabandoned; Ser. No. 853,781, filed Apr. 18, 1986, presently abandoned;Ser. No. 825,104, filed Jan. 31, 1986, presently abandoned; and Ser. No.882,051, filed Jul. 3, 1986, presently issued as U.S. Pat. No.5,002,887.

This invention relates to substances having tissue plasminogenactivator-type (t-PA) activity. More specifically, this inventionrelates to "recombinant" thrombolytic proteins, a process for obtainingthe proteins from genetically engineered cells, and the therapeutic useof the substances as thrombolytic agents.

These proteins are active thrombolytic agents which, it is contemplated,possess improved fibrinolytic profiles relative to native human t-PA.This may be manifested as increased affinity to fibrin, decreasedreactivity with inhibitors of t-PA, faster rate of thrombolysis,increased fibrinolytic activity and/or prolonged biological half-life.It is also contemplated that proteins of this invention can be moreconveniently prepared in more homogeneous form than can native humant-PA. An improved overall pharmacokinetic profile is contemplated forthese proteins.

The structure of native human t-PA can be viewed as comprising an amino(N-) terminus of about 91 amino acid residues, two so-called "kringle"regions, and at the carboxy terminus a serine protease-type domain. Wehave found that the N-terminus contains several sub-domains which playfunctional roles, inter alia, in fibrin binding and in the in vivoclearance of the protein. Recently the recovery of another form of t-PAwhich lacks the native N-terminus and first kringle region has beenreported, see European Published Patent Application No. 0 196 920(published 08 Oct. 1986). According to that report the truncated form oft-PA, which begins with Ala-160 of native human t-PA, isfibrinolytically active.

As described in greater detail hereinafter, this invention providesnovel protein analogs of human t-PA which retain both kringle regions ofnative human t-PA, but contain modifications within the N-terminus.While in certain embodiments the modifications involve deletions in theN-terminus, the first kringle region is left intact, and the N-terminaldeletion is never greater than 94 amino acids. Most embodiments involvesignificantly smaller deletion(s) and/or amino acid substituent(s). Byretaining more of the structure of native human t-PA, it is contemplatedthat the proteins of this invention selectively retain more of thedesirable biological activities of native human t-PA and may be lessimmunogenic than more drastically modified analogs of t-PA. It istherefore contemplated that the proteins of this invention possessimproved fibrinolytic and pharmacokinetic profiles relative to bothnative human t-PA and the truncated Ala-160 t-PA, as well as othermodified forms of t-PA.

The polypeptide backbone of natural human t-PA also includes fourconsensus Asn-linked glycosylation sites. It has been shown that two ofthese sites are typically glycosylated in t-PA from melanoma-derivedmammalian cells, i.e. at Adn₁₁₇ and Asn₄₄₈. Asn₁₈₄ is glycosylatedsometimes and Asn₂₁₈ is typically not glycosylated. t-PA frommelanoma-derived mammalian cells, e.g. Bowes cells, it also referred toherein as"native" or "natural" human t-PA.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the regions for amino acid deletions or substitutionswithin the present invention.

FIG. 2 shows exemplary modifications at N-linked glycosylation sites.

FIG. 3 shows illustrative proteins containing modification at Arg-275and at least one N-linked glycosylation site.

FIG. 4 shows illustrative proteins having N-terminal deletions. Theproteins have the peptide sequence shown in FIG. 3, wherein R1, R2 andR3 represent the wild-type tripeptide sequences, but wherein theN-termini (Gly-(-3) through Thr-91) are replaced with the sequencesshown.

FIG. 5 shows illustrative proteins containing a deletion of 1 to 94amino acids at the N-terminus and optional modification at either orboth of Arg-275 and at least one N-linked glycosylation site. (Forgeneral sequence, see FIG. 3).

FIG. 6 shows illustrative N-termini containing a deletion of 1-94 aminoacids. Specific proteins of this invention may be referred to by a3-part designation comprising a compound number from FIG. 5, followed bya designation of N-terminus and then identification of position 275. forexample, compound No. 2-11/N-6/Arg designates a protein wherein the 3glycosylation sites are deleted ("2-11", see FIG. 5), C-36 through C-43are deleted (N-terminus #N-6) and Arg-275 is retained.

FIGS. 7, 7A, 7B, and 7C exemplary proteins having a deletion of 1-45amino acids in the region from Ser-1 through Ser-50 and a modificationat either or both of Arg-275 and at least one N-linked glycosylationsite. (For general sequence, see FIG. 3). Illustrative proteins are asdefined in FIG. 5, but with the illustrated N-termini replacing the wildtype (wt) sequence of Gly-(-3) through Thr-91.

FIGS. 8, 8A and 8B show exemplary proteins having a deletion of 1-41amino acids from the region Cys-51 through Thr-91. (For generalsequence, see FIG. 3). Illustrative proteins are as defined in FIG. 5,but with the illustrated N-termini replacing the wild-type (wt) sequenceof Gly-(-3) through Thr-91.

FIG. 9A shows examples of proteins wherein the three glycosylation sitesand Arg-275 are deleted. FIG. 9B shows examples of proteins wherein noglycosylation sites are modified, but which contain various deletions.FIG. 9C shows compounds with deletions as above, which are also modifiedat the first N-linked glycosylation site, here by replacing Asn-117 withGln. FIG. 9D shows compounds containing deletions as above, in which allthree glycosylation sites are modified by replacement of Asn with Gln.

FIG. 10 shows exemplary proteins having one or more deletions of lessthan about 20 amino acids within the region Gly-(-3) through Thr-91.(For general purpose, see FIG. 3). Illustrative proteins are as definedin FIG. 5, but with the illustrated N-termini replacing the wild type(wt) sequence of Gly-(-3) through Thr-91.

FIG. 11 shows illustrative amino acid substitutions within the presentinvention.

FIGS. 12, 12A, 12B, 12C, and 12D show exemplary proteins containingsubstitutions for one or more amino acids within the region Gly-(-3)through Thr-91. (For general sequence, see FIG. 3). Illustrativeproteins are as defined in FIG. 5, but with the illustrated N-terminireplacing the wild type (wt) sequence of Gly-(-3) through Thr-91.

FIGS. 13, 13A and 13B show illustrative compounds with multipleN-terminal substitutions with Asn-117 replaced with Gln and Arg-275replaced with Thr.

FIG. 14 shows exemplary oligonucleotides for mutagenesis.

FIG. 15 shows exemplary mutagenized cDNA fragments.

FIGS. 16, 16A and 16B show methods of preparing illustrative compoundswithin the present invention.

This invention, as mentioned above, involves novel protein analogs ofhuman t-PA which possess t-PA-type thrombolytic activity. The proteinsof this invention differ in structure from human t-PA in that theycontain modifications in peptide sequence (i) at up to three of theAsn-linked glycosylation sites present in native t-PA; (ii) within theN-terminus of the proteins corresponding to the 94 amino acid matureN-terminus of native t-PA; and/or (iii) at the proteolytic cleavage sitespanning Arg-275 and Ile-276. These features of the proteins of thisinvention are described in grater detail below. Notwithstanding thevarious modifications, the numbering of amino acids as shown in theone-letter code sequence of Table 1 is retained.

A. Modifications at the N-terminus

In one aspect of this invention the proteins are characterized bydeletion of 1-94 amino acids within the peptide region spanning Gly-(-3)or Ser-1 through Thr-91, relative to native human t-PA. In oneembodiment, for example, Cys-51 through Asp-87 of native t-PA aredeleted. In two other specific embodiments Cys-6 through Ser-50, andCys-6 through Ile-86 are deleted, respectively. In other embodiments,more conservative modifications are present in the N-terminal region ofthe proteins. For instance, certain proteins of this invention containone or more amino acid deletions or substitutions within one or more ofthe following, more discrete subregions as shown in FIG. 1:

These and other modifications within the N-terminus spanning Gly-(-3)through Thr-91 are described in greater detail hereinafter.

B. Modifications at N-linked Glycosylation Sites

The protein variants of this invention may further contain no N-linkedcarbohydrate moieties or may be only partially glycosylated relative tonatural human t-PA. A"partially glycosylated" protein, as the phrase isused herein, means a protein which contains fewer N-linked carbohydratemoieties than does fully-glycosylated native human t-PA. This absence ofglycosylation or only partial glycosylation results from amino acidsubstitution or deletion at one or more of the concensus N-linkedglycosylation recognition sites present in the native t-PA molecule. Wehave found that variant proteins of this invention embodying suchmodification at one or more N-linked glycosylation sites retaint-PA-type thrombolytic activity with greater fibrinolytic activity incertain cases, may be more readily produced in more homogeneous formthan native t-PA, and in many cases have longer in vivo half-lives thannative t-PA.

N-linked glycosylation recognition sites are presently believed tocomprise tripeptide sequences which are specifically recognized by theappropriate cellular glycosylation enzymes. These tripeptide sequencesare either asparagine-X-threonine or asparagine-X-serine, where X isusually any amino acid. Their location within the t-PA peptide sequenceis shown in FIG. 3. A variety of amino acid substitutions or deletionsat one or more of the three positions of a glycosylation recognitionsite results in non-glycosylation at the modified sequence. By way ofexample, Asn₁₁₇ and Asn₁₈₄ of t-PA have both been replaced with Thr inone embodiment and with Gln in another embodiment. At least in the caseof the double Gln replacement, the resultant glycoprotein (Gln₁₁₇Gln₁₈₄) should contain only one N-linked carbohydrate moiety (at Asn₄₄₈)rather than two or three such moieties as in the case of native t-PA.Those skilled in the art will appreciate that analogous glycoproteinshaving the same Asn₄₄₈ monoglycosylation may be prepared by deletion ofamino acids or substitution of other amino acids at positions 117 and184 and/or by deleting or substituting one or more amino acids at otherpositions within the respective glycosylation recognitions sites, e.g.at Ser₁₁₉ and Ser₁₈₆, as mentioned above and/or by substitution, or morepreferably by deletion, at one or more of the"X" positions of thetripeptide sites. In another embodiment Asn at positions 117, 184 and448 are replaced with Gln. The resultant variants should contain noN-linked carbohydrate moieties, rather than two or three such moietiesas in the case of native t-PA. In other embodiments, potentialglycosylation sites have been modified individually, for instance byreplacing Asn, e.g. with Gln, at position 117 in one presently preferredembodiment, at position 184 in another embodiment and at position 448 instill another embodiment. This invention encompasses suchnon-glycosylated, monoglycosylated, diglycosylated and triglycosylatedt-PA variants.

Exemplary modifications at one or more of the three consensus N-linkedglycosylation sequences, R¹, R² and R³, as found in various embodimentsof this invention are depicted in FIG. 2:

C. Modification of the Arg-275/Ile-276 Cleavage Site

In one aspect of this invention the variants are optionally modified atthe proteolytic cleavage site spanning Arg-275 and Ile-276 by virtue ofdeletion of Arg-275 or substitution of another amino acid, preferably anamino acid other than Lys or His, for the Arg. Thr is at present anespecially preferred replacement amino acid for Arg-275 in the variousembodiments of this invention. Proteolytic cleavage at Arg-275 of nativet-PA yields the so-called"two-chain" molecule, as is known in the art.Proteins of this invention which are characterized by modification atthis cleavage site may be more readily produced in more homogeneous formthan the corresponding protein without the cleavage site modification,and perhaps more importantly may possess an improved fibrinolyticprofile and pharmacokinetic characteristic.

This invention thus provides a family of novel thrombolytic proteinsrelated to human t-PA. This family comprises several genera of proteins.

In one embodiment the proteins are characterized by a peptide sequencesubstantially the same as the peptide sequence of human t-PA, whereinArg-275 is deleted or is replaced by a different amino acid, preferablyother than lysine or histidine, and at least one of the consensusAsn-linked glycosylation sites is deleted or is modified to other than aconsensus Asn-linked glycosylation sequence. Exemplary proteins of thisembodiment are depicted in Table 1 below. By "characterized by a peptidesequence substantially the same as the peptide sequence of human t-PA,"as the phrase is used herein, we mean the peptide sequence of humant-PA, or a peptide sequence encoded by a DNA sequence encoding humant-PA or a DNA sequence capable of hybridizing thereto under stringenthybridization conditions. Thus the proteins of this invention includeanalogs of t-PA characterized by the various modifications orcombinations of modifications as disclosed herein, which may alsocontain other variations, e.g. allelic variations or additionaldeletion(s), substitution(s) or insertion(s) of amino acids which stillretain thrombolytic activity, so long as the DNA encoding those proteins(prior to the modification of the invention) is still capable ofhybridizing to a DNA sequence encoding human t-PA under stringentconditions.

In a second embodiment the proteins are characterized by a peptidesequence substantially the same as the peptide sequence of human t-PAwherein one or more amino acids are deleted within the N-terminal regionfrom Gly-(-3) through Thr-91 and wherein (a) one or more Asn-linkedglycosylation sites are optionally deleted or otherwise modified toother than a consensus Asn-linked glycosylation site, and/or (b) Arg-275is optionally deleted or replaced by a different amino acid, preferablyother than lysine or histidine. Exemplary proteins of this embodimentare shown in FIG. 4:

This embodiment includes a subgenus of proteins wherein 1 to about 94amino acids are deleted from the region Gly-(-3) through Thr-91 and oneor more of the Asn-linked glycosylation sites are deleted or otherwisemodified to other than a consensus Asn-linked glycosylation sequence aspreviously described. Also included is a subgenus of compounds wherein 1to about 94 amino acids are deleted from the region Gly-(-3) throughThr-91, and Arg-275 is deleted or replaced with a different amino acid,preferably other than lysine or histidine. A further subgenus of thisembodiment is characterized by a deletion of 1 to about 94 amino acidsfrom within the regions Gly-(-3) through Thr-91, deletion ormodification of one or more of the Asn-linked glycosylation sites (seee.g. the table on page 6) and deletion of Arg-275 or replacement thereofwith a different amino acid. Exemplary proteins of these subgenera aredepicted in FIGS. 5 and 6.

This embodiment also includes a subgenus of proteins wherein theN-terminal deletion comprises a deletion of 1 to about 45 amino acidsfrom within the region Ser-1 through Ser-50. Also included is a subgenusof proteins wherein 1 to about 45 amino acids are deleted from withinthe region Ser-1 through Ser-50 and one or more glycosylation sites aremodified as previously described. A further subgenus comprises proteinshaving a deletion of 1 to about 45 amino acids from within the regionSer-1 through Ser-50, wherein Arg-275 is deleted or replaced withanother amino acid. Additionally included is a subgenus having deletionof 1 to about 45 amino acids from within the region Ser-1 through Ser-50and wherein both of (a) one or more glycosylation sites, and (b)Arg-275, are optionally modified as previously described. Exemplaryproteins of these subgenera are depicted in FIG. 7, below, as well as inFIGS. 5 and 6.

With reference to the modified N-termini depicted in FIG. 7, it shouldbe understood that more than one amino acid may be deleted. Wheremultiple amino acids are deleted, they may be adjacent to one another,or separated by one or more other amino acids. In designating compoundswith such N-termini we indicate the size of the deletion by"Δn" where"n" is the number of amino acids deleted. For example, N-terminus #24wherein one amino acid is deleted as shown in FIG. 7 may be referred toas N-24Δ1". Where two amino acids are deleted, e.g. S-1 and Y-2, theN-terminus is referred to as"N-24Δ2", etc. Where combinations ofdeletions are made, e.g. wherein S-1, Y-2 and I-5 are deleted, theN-terminus may be referred to as"N-24Δ2, N-28Δ1". As indicated in thetext following FIG. 6, specific compounds are designated by a 3-partcode comprising a compound number from FIG. 5 followed by a designationof N-terminus #, e.g. from FIG. 6, and then identification of the statusof position 275. Compound 2-26/N-24Δ2, N-28Δ1/- thus designates theprotein wherein all three glycosylation sites; R-275; S-1, Y-2 and I-5are deleted.

This embodiment further includes a subgenus of proteins wherein 1 toabout 41 amino acids are deleted from the region Cys-51 through Thr-91.Proteins of this subgenus may optionally be modified such that Arg-275is deleted or replaced with a different amino acid, preferably otherthan lysine or histidine. Proteins of this subgenus may, as analternative to or in addition to the modification at Arg-275, bemodified such that (a) one or more N-linked glycosylation sites areabolished, as previously described and/or (b)one or more amino acids aredeleted within the region Gly-(-3) through Ser-50. Exemplary proteins ofthis subgenus are similar to those depicted in FIGS. 5 and 7, butcontain N-termini such as those depicted in FIG. 8, below.

With reference to FIG. 8, above, it should be noted that severalsubclasses of proteins are disclosed. For example, proteins containing adeletion of 1-37 amino acids (individually, consecutively, or incombination) from Cys-51 through Asp-87 are depicted, as are proteinscontaining a deletion of 1-37 amino acids from Cys-51 through Arg-87 anda deletion of one or more amino acids within the region Gly-(-3) throughCys-51 (See N-76). With reference to compounds containing an N-terminusselected from N-termini N-76 through N-111 it should be understood thatmore than one amino acid may be deleted. For example, N-77 wherein oneamino acid is deleted, as shown in FIG. 8, may be referred toas"N-77Δ1". Where six amino acids are deleted, e.g. S-52 through F-57,the N-terminus is referred to as"N-77Δ6", etc. Specific proteins aredesignated as described following FIGS. 5 and 7. Examples wherein thethree glycosylation sites and Arg-275 are deleted are shown in FIG. 9A:

Illustrative compounds wherein no modifications are present atglycosylation sites, but which contain various deletions are shown inFIG. 9B:

Illustrative compounds with the same deletions as above, but which arealso modified at the first N-linked glycosylation site, here byreplacing Asn-117 with Gln, are listed in FIG. 9C:

Illustrative compounds with the same deletions as above, but which arealso modified at all three glycosylation sites, here by replacement ofAsn's with Gln's, are listed in FIG. 9D:

Thus, this embodiment further includes a subgenus of proteins whereinone or more deletions of less than about 20 amino acids are presentwithin the region Gly-(-3) through Thr-91. Proteins of this subgenus mayalso be modified at Arg-275 and/or at one or more of the Asn-linkedglycosylation sites. Exemplary proteins of this subgenus are similar tothose depicted in FIGS. 5 through 8, but contain in place of the wildtype N-terminus, an N-terminus such as those depicted in FIG. 10, below.Additional exemplary compounds of this subgenus are also listed above bytheir 3-part code designations.

In a third embodiment the proteins are characterized by a peptidesequence substantially the same as the peptide sequence of human t-PAwherein different amino acids are substituted for 1-94 of the aminoacids in the region Gly-(-3) through Thr-91. This embodiment includes asubgenus of compounds characterized by replacement of one or more aminoacids within the above-mentioned N-terminus and by modification atArg-275 as previously described. Also included is a subgenus ofcompounds characterized by the above-mentioned replacement of one ormore amino acids within the N-terminus and modification, as previouslydescribed, at one or more of the consensus Asn-linked glycosylationsites. A further subgenus of this embodiments is characterized bysubstitution of one or more amino acids within the N-terminus, andmodifications as previously described, at both Arg-275 and at one ormore of the N-Linked glycosylation sites. In one aspect of thisembodiment the amino acid substitution(s) is/are within the regionGly-(-3) through Ser-50, with or without modification at Arg-275 and/orone or more of the N-linked glycosylation sites. In another aspect, theamino acid substitution(s) is/are within the region Cys-51 throughThr-91, again, with or without modification at Arg-275 and/or at one ormore of the N-linked glycosylation sites. In a further aspect, one toabout eleven, preferably one to about 6 amino acids are replaced withinone or more of the following regions, again with or without the otherabove-mentioned modification(s) as shown in FIG. 1:

In a further aspect of this embodiment, the substitution(s) is/arepresent in one or more of the following regions: R-7 through S-20, W-21through Y-33, N-37 through Q-42, and H-44 through S-50. In an additionalrespect of this embodiment, the N-terminus is modified, again, bysubstitution for one to about eleven, preferably one to about six, aminoacids in one or more of the above defined regions, and is furthermodified by deletion of one to 93, preferably 1 to about 45, and morepreferably 1 to about 15, amino acids.

Illustrative amino acid substitutions are shown in FIG. 1, below, andexemplary proteins are depicted in FIG. 12. Of the replacement aminoacids for R-40, A-41 and Q-42, presented in FIG. 11, S is a preferredreplacement for R-40, and V and L are preferred replacements for A-41and Q-42, respectively. It should be noted that proteins of thisinvention embodying the substitutions identified for R-40, A-41 and Q-42in FIG. 11 are presently preferred, alone, or, as in other aspects andsubgenera of this embodiment, in combination with other substitution(s)and/or deletions within the N-terminus, and/or modifications at R-275and/or at at least one glycosylation site. It is contemplated that tothe extent that our proteins are modified by substitution rather thandeletion, our proteins

Proteins of this invention embodying amino acid substitution(s) may bedesignated by a 3-part code as previously described. It should beunderstood of course that more than one wt amino acid may be replaced.In designating compounds with such N-termini we indicate the number ofsubstitutions by"sn" where "n" is the number of amino acids replaced,e.g with the replacement amino acids such as (but not limited to) thosedepicted in FIG. 11. For example, N-terminus #N-122s1 designatesN-terminus 122 as depicted in FIG. 12, while N-terminus #N-122s4designates that N-terminus wherein S-1 is replaced with G and thefollowing three wt amino acids are replaced with other amino acids.Proteins of this embodiment containing multiple amino acid substitutionsmay be designated by a string of N-terminus designations indicatingspecific replacements, as shown in FIG. 13:

One subgenus of particular interest is characterized by replacement ofone or more of Y-67 through S-69, with optional deletion of, and/orsubstitution for, one or more amino acids from Gly-(-3) through L-66,with or without modification as described above at one or moreglycosylation sites and/or at Arg-275.

In one aspect of the invention the proteins contain at least oneso-called"complex carbohydrate" sugar moiety characteristic of mammalianglycoproteins. As exemplified in greater detail below, such"complexcarbohydrate" glycoproteins may be produced by expression of a DNAmolecule encoding the desired polypeptide sequence in mammalian hostcells. Suitable mammalian host cells and methods for transformation,culture, amplification, screening, and product production andpurification are known in the art. See e.g. Gething and Sambrook, Nature293:620-625 (1981), or alternatively, Kaufman et al., Molecular andCellular Biology 5 (7):1750-1759 (1985) or Howley et al., U.S. Pat. No.4,419,446.

A further aspect of this invention involves t-PA variants as definedabove in which each carbohydrate moiety is a processed form of theinitial dolicol-linked oligosaccharide characteristic of insectcell-produced glycoproteins, as opposed to a"complex carbohydrate"substituent characteristic of mammalian glycoproteins, includingmammalian derived t-PA. Such insect cell-type glycosylation is referredto herein as"high mannose" carbohydrate for the sake of simplicity. Forthe purpose of this disclosure, complex and high mannose carbohydratesare as defined in Kornfield et al., Ann. Rev. Biochem. 54: 631-64(1985)."High mannose" variants in accordance with this invention arecharacterized by a variant polypeptide backbone as described above whichcontains at least one occupied N-linked glycosylation site. Suchvariants may be produced by expression of a DNA sequence encoding thevariant in insect host cells. Suitable insect host cells as well asmethods and materials for transformation/transfection, insect cellculture, screening and product production and purification useful inpracticing this aspect of the invention are known in the art.Glycoproteins so produced also differ from natural t-PA and from t-PAproduced heretofore by recombinant engineering techniques in mammaliancells in that the variants of this aspect of the invention do notcontain terminal sialic acid or galactose substituents on thecarbohydrate moieties or other protein modifications characteristic ofmammalian derived glycoproteins.

The proteins of this invention which contain no N-linked carbohydratemoieties may also be produced by expressing a DNA molecule encoding thedesired variant, e.g. compounds 1-6 through 1-11 of FIG. 3, inmammalian, insect, yeast or bacterial host cells, with eucaryotic hostcells being presently preferred. As indicated above suitable mammalianand insect host cells, and in addition, suitable yeast and bacterialhost cells, as well as methods and materials fortransformation/transfection, cell culture, screening and productproduction and purification useful in practicing this aspect of theinvention are also known in the art.

Additionally, as should be clear to those of ordinary skill in this art,this invention also contemplates other t-PA variants which arecharacterized, instead of by amino acid deletion within the region Gly₋₃or Ser₁ through Thr₉₁, by one or more amino acid substitutions withinthat region, especially in the region Arg₇ through Ser₅₀, or by acombination of deletion and substitution. cDNAs encoding these compoundsmay be readily prepared, e.g., by methods closely analogous to themutagenesis procedures described herein using appropriate mutagenesisoligonucleotides. The cDNAs may be optionally mutagenized at one or moreof the codons for R¹, R² and R³, and/or Arg-275, and may be insertedinto expression vectors and expressed in host cells by the methodsdisclosed herein. It is contemplated that these proteins will share theadvantageous pharmacokinetic properties of the other compounds of thisinvention, and perhaps avoid undue antigenicity upon administration inpharmaceutical preparations analogous to those disclosed herein.

As should be evident from the preceding, all variants of this inventionare prepared by recombinant techniques using DNA sequences encoding theanalogs which may also contain fewer or no potential glycosylation sitesrelative to natural human t-PA and/or deletion or replacement ofArg-275. such DNA sequences may be produced by conventionalsite-directed mutagenesis of DNA sequences encoding t-PA.

DNA sequences encoding t-PA have been cloned and characterized. Seee.g., D. Pennica et al., Nature (London) 301:214(1983) and R. Kaufman etal., Mol. Cell. Biol.5(7):1750 (1985). One clone, ATCC 39891, whichencodes a thrombolytically active t-PA analog is unique in that itcontains a Met residue at position 245 rather than Val. Typically, theDNA sequence encodes a leader sequence which is processed, i.e.,recognized and removed by the host cell, followed by the amino acidresidues of the full length protein, beginning withGly.Ala.Arg.Ser.Tyr.Gln. . . Depending on the media and host cell inwhich the DNA sequence is expressed, the protein so produced may beginwith the Gly.Ala.Arg amino terminus or be further processed such thatthe first three amino acid residues are proteolytically removed. In thelatter case, the mature protein has an amino terminus comprising:Ser.Tyr.Gln.Leu . . . t-PA variants having either amino terminus arethrombolytically active and are encompassed by this invention. Variantsin accord with the present invention also include proteins having eitherMet₂₄₅ or Val₂₄₅, as well as other variants, e.g. allelic variations orother amino acid substitutions or deletions, which still retainthrombolytic activity.

This invention also encompasses compounds as described above whichcontain a further modification in the polypeptide domain spanningAsn-218 through Thr-220. Specifically, compounds of this embodiment arefurther characterized by an amino acid other than Asn or a peptide bondat position 218 and/or an amino acid other than Pro or a peptide bond atposition 219 and/or an amino acid other than Ser or Thr or a peptidebond at position 220. Compounds of this embodiment thus lack theconsensus N-linked glycosylation site which is typically notglycosylated in t-PA produced by melanoma-derived mammalian cells.

As mentioned above, DNA sequences encoding individual variants of thisinvention may be produced by conventional site-directed mutagenesis of aDNA sequence encoding human t-PA or analogs or variants thereof. Suchmethods of mutagenesis include the M13 system of Zoller and Smith,Nucleic Acids Res. 10:6487-6500 (1982); Methods Enzymol. 100: 468-500(1983); and DNA 3:479-488 (1984), using single stranded DNA and themethod of Morinaga et al., Bio/technology, 636-639 (July 1984), usingheteroduplexed DNA. Several exemplary oligonucleotides used inaccordance with such methods to effect deletions in the N-terminus or toconvert an asparagine residue to threonine or glutamine, for example,are shown in FIG. 14. It should be understood, of course, that DNAencoding each of the glycoproteins of this invention may be analogouslyproduced by one skilled in the art through site-directed mutagenesisusing(an) appropriately chosen oligonucleotide(s). Expression of the DNAby conventional means in a mammalian, yeast, bacterial, or insect hostcell system yields the desired variant. Mammalian expression systems andthe variants obtained thereby are presently preferred.

The mammalian cell expression vectors described herein may besynthesized by techniques well known to those skilled in this art. Thecomponents of the vectors such as the bacterial replicons, selectiongenes, enhancers, promoters, and the like may be obtained from naturalsources or synthesized by known procedures. See Kaufman et al., J. MolBiol., 159:51-521 (1982); Kaufman, Proc Natl. Acad. Sci. 82:689-693(1985).

Established cell lines, including transformed cell lines, are suitableas hosts. Normal diploid cells, cell strains derived from in vitroculture of primary tissue, as well as primary explants (includingrelatively undifferentiated cells such as hematopoetic stem cells) arealso suitable. Candidate cells need not be genotypically deficient inthe selection gene so long as the selection gene is dominantly acting.

The host cells preferably will be established mammalian cell lines. Forstable integration of the vector DNA into chromosomal DNA, and forsubsequent amplification of the integrated vector DNA, both byconventional methods, CHO (Chinese hamster Ovary) cells are presentlypreferred. Alternatively, the vector DNA may include all or part of thebovine papilloma virus genome (Lusky et al., Cell, 36:391-401 (1984) andbe carried in cell lines such as C127 mouse cells as a stable episomalelement. Other usable mammalian cell lines include but are not limitedto, HeLa, Cos-1 monkey cells, mouse L-929 cells, 3T3 lines derived fromSwiss, Balb-c or NIH mice, BHK or HaK hamster cells lines and the like.

Stable transformants then are screened for expression of the product bystandard immunological or enzymatic assays. The presence of the DNAencoding the variant proteins may be detected by standard proceduressuch as Southern blotting. Transient expression of the DNA encoding thevariants during the several days after introduction of the expressionvector DNA into suitable host cells such as COS-1 monkey cells ismeasured without selection by activity or immunologic assay of theproteins in the culture medium.

In the case of bacterial expression, the DNA encoding the variant may befurther modified to contain different codons for bacterial expression asis known in the art and preferably is operatively linked in-frame to anucleotide sequence encoding a secretory leader polypeptide permittingbacterial expression, secretion and processing of the mature variantprotein, also as is known in the art. The compounds expressed inmammalian, insect, yeast or bacterial host cells may then be recovered,purified, and/or characterized with respect to physicochemical,biochemical and/or clinical parameters, all by known methods.

These compounds have been found to bind to monoclonal antibodiesdirected to human t-PA, and may thus be recovered and/or purified byimmunoaffinity chromatography using such antibodies. Furthermore, thesecompounds possess t-PA-type enzymatic activity, i.e., compounds of thisinvention effectively activate plasminogen in the presence of fibrin toevoke fibrinolysis, as measured in an indirect assay using the plasminchromogenic substrate S-2251 as is known in the art.

This invention also encompasses compositions for thrombolytic therapywhich comprise a therapeutically effective amount of a variant describedabove in admixture with a pharmaceutically acceptable parenteralcarrier. Such composition can be used in the same manner as thatdescribed for human t-PA and should be useful in humans or lower animalssuch as dogs, cats and other mammals known to be subject to thromboticcardiovascular problems. It is contemplated that the compositions willbe used both for treating and desirably for preventing thromboticconditions. The exact dosage and method of administration will bedetermined by the attending physician depending on the potency andpharmacokinetic profile of the particular compound as well as on variousfactors which modify the actions of drugs, for example, body weight,sex, diet, time of administration, drug combination, reactionsensitivities and severity of the particular case.

The following examples are given to illustrate embodiments of theinvention. It will be understood that these examples are illustrative,and the invention is not to be considered as restricted thereto exceptas indicated in the appended claims.

In each of the examples involving insect cell expression, the nuclearpolyhedrosis virus used was the L-1 variant of the AutographaCalifornica, and the insect cell line used was the spodoptera frugiperdaIPLB-SF21 cell line (Vaughn, J. L. et al., In Vitro (1977) 13, 213-217).The cell and viral manipulations were as detailed in the literature(Pennock G. D., et al., supra; Miller, D. W., Safer, P., and Miller, L.K., Genetic Engineering, Vol. 8, pages 277-298, J. K. Setlow and A.Hollaender, eds. Plenum Press, 1986). The RF m13 vectors, mp18 and mp11, are commercially available from New England Biolabs. However, thoseof ordinary skill in the art to which this invention pertains willappreciate that other viruses, strains, host cells, promoters andvectors containing the relevant cDNA, as discussed above, may also beused in the practice of each embodiment of this invention. The DNAmanipulations employed are, unless specifically set forth herein, inaccordance with Maniatis et al., Molecular Cloning: A Laboratory Manual(Cold Spring Harbor, N.Y. 1982).

Plasmid Derivations

Mutagenesis of cDNAs at codons for the various amino acids was conductedusing an appropriate restriction fragment of the cDNA in M13 plasmids bythe method of Zoller and Smith. Deletions within the cDNA were effectedby loopout mutagenesis using an appropriate restriction fragment, e.g.the SacI fragment, of the cDNA either in M13 vectors or by heteroduplexloop-out in plasmid pSVPA4.

The plasmid pSVPA4 was constructed to allow the expression of t-PAglycoprotein in mammalian cells. This plasmid was made by first removingthe DNA encoding the SV40 large T polypeptide from the plasmid pspLT5(Zhu, Z. et al., 1984, J. Virology 51:170-180). This was accomplished byperforming a total Xho 1 digest followed by partial Bam-Hl restrictionendonuclease digestion. The SV40 large T encoding region in pspLT5 wasreplaced with human t-PA-encoding sequence by ligating a cohesiveSall/BamHl t-PA encoding restriction fragment, isolated by digestingplasmid J205 (ATCC No. 39568) with Sal I and BamHl, to the parentXhol/BamHl cut vector pspLT5 prepared as described above. Consequently,t-PA will be transcribed in this vector under the control of the SV40late promoter when introduced into mammalian cells. This final contructis designated pSVPA4.

Plasmid pLDSG is an amplifiable vector for the expression of t-PA inmammalian cells such as CHO cells. pLDSG contains a mouse DHFR cDNAtranscription unit which utilizes the adenovirus type 2 major latepromoter (MLP), the simian virus 40 (SV40) enhancer and origin ofreplication, the SV40 late promoter (in the same orientation as theadenovirus MLP), a gene encoding tetracyclin resistance and a cDNAencoding human t-PA (Met-245) in the proper orientation with respect tothe adenovirus type 2 MLP. The preparation of pLDSG from pCVSVL2 (ATCCNo. 39813) and a t-PA encoding cDNA has been described in detail as hascotransformation with, and amplification of, pLDSG in CHO cells. Kaufmanet al., Mol. and Cell. Bio. 5(7): 1750-1759 (1985).

Plasmid pWGSM is identical to pLDSG except that the cDNA insert encodesMet-245 human t-PA. pWSGM may be constructed using cDNA from plasmidJ205 (ATCC No. 39568) or pIVPA/1 (ATCC No. 39891). Throughout thisdisclosure pWGSM and pLDSG may be used interchangeably, although asindicated previously, the former vector will produce Val-245 proteinsand the latter Met-245 proteins.

pIVPA/1 (ATCC No. 39891) is a baculoviral transplacement vectorcontaining a t-PA-encoding cDNA. pIVPA/1 and mutagenized derivativesthereof are used to insert a desired cDNA into a baculoviral genome suchthat the cDNA will be under the transcriptional control of thebaculoviral polyhedrin promoter.

Heteroduplex Mutagenesis

The mutagenesis via heteroduplexed DNA of specific areas in the t-PAexpression plasmid, pSVPA4, involves the following steps: Preparation ofampicillin sensitive pSVPA4 DNA

1. Plasmid pSVPA4 (15 ug) was linearized with PvuI to completion. Thismixture was extracted with phenol/chloroform and the DNA wasprecipitated using two volumes of ethanol with 0.1M NaCl present.

2. The DNA was resuspended in a 21 ul of water, 1 ul dNTB solution(containing 2 mM dATP, dGTP, dTTP, dCTP), 2.5 ul 10× nick translationbuffer (0.5M Tris-Cl pH 7.5, 0.1M MgSO₄, 10 mM DTT, 500 ug/ml) and 0.5ul (2 units) DNA polymerase 1-Large Fragment (New England Biolabs). Thismixture was incubated at room temperature for thirty minutes and thenphenol/chloroform extracted followed by ethanol precipitation asdescribed above.

3. The precipitated DNA was resuspended to 0.2 ug/ul by the addition of75 ul water.

Preparation of ampicillin resistant pSVPA4 DNA

1. Plasmid pSVPA4 (15 ug) was digested with Sac I which cuts thisplasmid twice within the t-PA encoding sequence to produce tworestriction fragments, a 1.4 kbp t-PA encoding restriction fragment plusthe parent vector. Following restriction digestion 1 ul (28 units) ofcalf intestine alkaline phospatase (Boehringer Mannheim) was added thenincubated at 37° C. for five minutes. The two bands were separated byloading this mixture onto a 0.7% agarose gel. The parent vectorrestriction fragment was excised from the gel and extracted byadsorption to silica dioxide at 4° C., which was followed by elution in50 mM Tris/1 mM EDTA at 37° C. for thirty minutes. The eluted DNA wasadjusted to a final concentration of 0.2 ug/ul.

Heteroduplex Annealing

1. Mix 6 ul (1.2 ug) of ampicillin sensitive pSVPA4 DNA with 6 ul (1.2ug) ampicillin resistant pSVPA4 DNA.

2. Add an equal volume (12 ul) of 0.4M NaOH. Incubate at roomtemperature for ten minutes.

3. Slowly add 4.5 volumes (108 ul) of 0.1M Tris-Cl pH 7.5/20 mM HCl.

4. 50 picomoles (5 ul) of phosphorylated mutagenic oligonucleotide wasadded to 45 ul of heteroduplex mixture.

5. This mixture was incubated at 68° C. for two hours then slowly cooledto room temperature.

Mutagenesis

1. Each mutagenesis reaction was adjusted to the followingconcentrations by the addition of 7 ul to the heteroduplex mixtures, 2mM MgCl/0.2 mM ATP/60 uM dATP, dTTP,dGTP,dCTP/4 mM DTT/40 units/mlKlenow fragment of E. coli DNA polymerase I (B.R.L.), 2000 units/ml T4DNA ligase (N.E.B.). This mixture was incubated at room temperature for2 hours.

2. The reaction was then phenol/chloroform extracted which was followedby ethanol precipitation. The precipitated DNA was resuspended in 12 ul50 mM Tris-Cl/1 mM EDTA. 4 ul of this was used to transform competentHB101 bacteria.

3. Ampicillin resistant colonies were screened with 1×10⁶ cpm/ml of a ³²P-labeled screening oligonucleotide in 5× SSC, 0.1% SDS, 5×denhardt'sreagent, and 100 ug/ml denatured salmon sperm DNA.

4. The filters were washed with 5× SSC, 0.1% SDS at a temperature 5°below the calculated melting temperature of the oligonucleotide probe.

5. DNA was prepared from positively hybriding clones and analyzedinitially by digestion with different restriction enzymes and agarosegel electrophoresis. DNA was transferred to nitrocellulose and filterswere prepared and hybridized to the screening probes in order to ensurethe mutagenic oligonucleotide was introduced in to the correct fragment.

6. DNA was then retransformed into E. coli and ampicillin resistantcolonies were screened for hybridization to the screeningoligonucleotide.

7. Final mutations were confirmed by DNA sequencing (Sanger).

Preparation of Mutagenized cDNAs: M13 method

The following schematic restriction map illustrates a cDNA encodinghuman t-PA (above) with cleavage sites indicated for specificendonucleases (indicated below): ##STR1## The initiation codon, ATG, andthe cDNA regions encoding (a), R¹, R² and R³ are indicated. Thus,mutagenesis at the N-terminus may be effected using the SacI fragment orthe BglII/NarI fragment, for example. Mutagenesis at Arg-275 and/or atR¹ and/or R² may be effected using, e.g., the SacI fragment orBglII/SacI fragment. Mutagenesis at R³ may be effected using, anEcoRI/XmaI or EcoRI/ApaI fragment. The choice of restriction fragmentmay be determined based on the convenience of using particular vectorsfor mutagenesis and/or for expression vector construction.

Generally, the cDNA restriction fragment to be mutagenized may beexcised from the full-length cDNA present, e.g., in pWGSM, pIVPA/1 orpSVPA4, using the indicated endonuclease enzyme(s) and then mutagenized,e.g. with the oligonucleotides shown in Table 7 or otheroligonucleotides designed for the desired mutagenesis.

Exemplary mutagenized cDNA fragments which may thus be prepared areshown in FIG. 15, below.

Following mutagenesis the fragment, with or without further mutagenesis,may then be excised from the M13 vector and ligated back into anexpression vector containing the full-length or partial cDNA previouslycleaved with the same enzyme(s) as were used for excising themutagenized fragment from the M13 vector. By this method the full-lengthcDNA, mutagenized as desired, may be re-assembled using one or moremutagenized fragments as restriction fragment cassettes.

cDNAs encoding the following illustrative compounds (see FIGS. 4 through7) may be prepared from the mutagenized fragments of FIG. 15 as shown inFIG. 16:

Plasmids pIVPA or pSVPA4, in addition to utility as expression vectors,may also be used as a"depot" in the construction of cDNAs having anydesired permutation of mutagenized sites. Thus,"pIVPA/Δ" or"pSVPA4/Δ",mutagenized (via M13 or heteroduplexing) plasmids containing a desiredmodification in the cDNA region encoding the N-terminal region may bedigested with NarI (partial) and XmaI (SmaI) (total) to remove the cDNAregion encoding the protein domain spanning R¹, R² and R³. A secondpIVPA or pSVPA4 plasmid mutagenized, if desired (via M13 orheteroduplexing), at any combination of Arg-275, R¹, R² and R³ -encodingregions may then be digested with NarI (total) and XmaI (SmaI) (total)and the NarI/XmaI (SmaI) fragment may then be identified, isolated andligated into the NarI/XmaI (SmaI) digested pIVPA/Δ or pSVPA4/Δ. Such useof the NarI/XmaI (SmaI) restriction fragment cassette, for example,allows the construction of desired mutagenized cDNAs in pIVPA or pSVPA4.The mutagenized cDNA may then be transferred, e.g. as a BglII/XmaIrestriction fragment cassette into BglII/XmaI-digested pWGSM formammalian expression, if desired.

EXAMPLES EXAMPLE 1 Preparation of Gln₁₁₇ Deletions Variants A.Preparation of Gln-117 truncated cDNA

cDNA molecules encoding the polypeptide sequence of compounds2-1/N-21/Arg, 2-1/N-22/Arg and 2-1/N-23/Arg were prepared using theoligonucleotide-directed mutagenesis method of Zoller and Smith.Specifically, the mutagenesis vector RF M13/t-PA containing the t-PAgene was constructed from the mammalian t-PA expression plasmid pSVPA4.RF M13/t-PA was constructed by first digesting pSVPA4 to completion withthe restriction endonuclease SacI. The approximately 1,436 base pair(bp) SacI fragment encodes a large portion of the polypeptide sequenceof t-PA and includes the nucleotide sequences encoding the consensusN-linked glycosylation sites encompassing asparagines 117,184 and 218.This 1,436 bp (hereinafter 1.4 kbp) fragment was purified by preparativeagarose gel electrophoresis.

The Sac I fragment of the t-PA cDNA, obtained as a SacI fragment, above,was ligated to a linearized double-stranded RF M13mp18 DNA vector whichhas been previously digested with Sac I. The ligation mixture was usedto transform transformation competent bacterial JM101 cells. M13 plaquescontaining t-PA-derived DNA produced from transformed cells wereidentified and isolated by analytical DNA restriction analysis and/orplaque hybridization. Radiolabeled oligo-nucleotides (˜17mers, ofpositive polarity) derived from within the SacI restriction sites of thet-PA-encoding nucleotide sequence depicted in FIG. 3 were used as probeswhen filter hybridization was employed to detect viral plaquescontaining tPA DNA. All oligonucleotides were prepared by automatedsynthesis with an Applied Biosystems DNA synthesizer according to themanufacturer's instructions.

Several of the positive plaques detected by restriction or hybridizationanalysis were then further cloned by conventional plaque purification.Purified M13/t-PA bacteriophage obtained from the plaque purificationprocedure was used to infect JM101 cells. These infected cells producecytoplasmic double-stranded"RF" M13t-PA plasmid DNA. The infected cellsalso produce bacteriophage in the culture medium which containsingle-stranded DNA complimentary to the 1.4 kbp SacI fragment of t-PAand to M13 DNA. Single-stranded DNA was purified from theM13/t-PA-containing phage isolated from the culture medium. Thissingle-stranded M13/t-PA DNA was used as a template in a mutagenesisreaction according to the method of Zoller and Smith usingoligonucleotide #3 of FIG. 14. This mutagenesis event changes the Asncodon to a Gln codon at position 117 of the subsequently obtained codingstrand of DNA by changing the DNA sequence from"AAC" to"CAG". Followingthe mutagenesis reaction, the DNA was transformed into the bacterialstrain JM 101. To identify mutagenized cDNA's, the transformant plaqueswere screened by DNA hybridization using radiolabeled oligonucleotide #4of FIG. 14. All exemplary oligonucleotides in FIG. 14 are of positivepolarity, i.e., represent portions of a coding rather than non-codingstrand of DNA. All hybridization positive plaques were further purifiedby subsequent secondary infections of JM 101 cells with M13 phagecontaining mutagenized DNA.

RF M13/t-PA plasmid DNA was purified from JM 101 cells infected withpurified M13 phage containing mutagenized t-PA cDNA. The RF M13/t-PAplasmid thus obtained contains the Gln₁₁₇ mutagenized Sac I restrictionfragment of t-PA DNA. This mutagenized restriction fragment can then befurther mutagenized, again by the method of Zoller and Smith, but usingthe oligonucleotides described below. The oligonucleotides describedbelow were designed to induce a deletion ("loop out") within the cDNAregion encoding the N-terminal domain.

Deletion Mutagenesis 1: Oligonucleotide #8 of FIG. 14 induce a cDNAdeletion encoding Cys-6 through Ser-50, inclusive. Following this secondmutagenesis reaction the DNA is transformed into JM 101 cells. Toidentify mutagenized cDNAs, the transformant plaques were screened asabove, but using radiolabeled oligonucleotide #9 of FIG. 14.Hybridization positive plaques can be further purified by subsequentsecondary infections of JM 101 cells with M13 phage containing the twicemutagenized t-PA cDNA. The cDNA prepared as described below whichcontains this mutagenized restriction fragment encodes compound2-1/N-21/Arg in which Ile-5 is covalently bonded to Cys-51 by a peptidebond.

Deletion Mutagenesis 2: Oligonucleotide #10 of FIG. 14 induced a cDNAdeletion encoding Cys₆ through Ile₈₆, inclusive. Following this secondmutagenesis reaction the DNA is transformed into JM 101 cells. Toidentify mutagenized cDNAs, the transformant plaques were screened asabove, but using radiolabeled oligonucleotide #11 of FIG. 14.Hybridization positive plaques can be further purified by subsequentsecondary infections of JM 101 cells with M13 phage containing the twicemutagenized t-PA cDNA. The cDNA prepared as described below whichcontains this mutagenized fragment encodes compound 2-1/N-22/Arg inwhich Ile₅ is covalently bonded to Asp₈₇ by a peptide bond.

Deletion Mutagenesis 3: Oligonucleotide #12 of FIG. 14 can be used togenerate a cDNA deletion encoding Cys₅₁ through Asp₈₇, inclusive.Following this second mutagenesis reaction the DNA is transformed intoJM 101 cells. To identify mutagenized cDNAs, the transformant plaqueswere screened as above, but using radiolabeled oligonucleotide #13 ofFIG. 14. Hybridization positive plaques can be further purified bysubsequent secondary infections of JM 101 cells with M13 phagecontaining the twice mutagenized t-PA cDNA. The cDNA prepared asdescribed below which contains this mutagenized restriction fragmentencodes compound 2-1/N-23/Arg in which Ser₅₀ is covalently bonded toThr₈₈ by a peptide bond.

Each of these mutagenized restriction fragments can then be ligated backinto the mammalian expression vector pSVPA4 as a SAC I cassette bymethods analogous to those described in Example #3B, or prepared forinsertion into the insect cell expression vector pIVPA/1 (ATCC No.39891) as a Bgl II/Sac I cassette derived from modified RF M13/t-PA DNA.

B. Preparation of Vectors Used for Expression of High Mannose Gln₁₁₇Deletion Variants

The purified RF M13/t-PA containing the modified and truncated t-PAcDNA, prepared as described above, can be digested with the restrictionendonucleases BglII and Sac I. The approximately 1.2 kbp BglII/Sac Irestriction fragment was purified by conventional preparative gelelectrophoresis. The BglII/Sac I fragment so obtained constitutes amutagenized cassette which lacks a 5' and 3' portion of the DNA whichencodes the amino and carboxy termini of the translated protein.

Insect expression vector pIVPA/1 (ATCC No. 39891) contains a wild typetPA cDNA insert operatively linked to a polyhedrin promoter togetherwith baculovirus flanking DNA sequences. pIVPA/1 was digested with BglIIand Sac I thereby excising a t-PA coding region spanning the N-terminusand R¹ and R². The BglII/Sac I cassettes containing the mutagenized,N-terminally modified t-PA cDNA fragments may each then be ligated topIVPA/1 expression vector DNA which had been previously purifiedfollowing digestion with BglII and SacI. The resulting plasmids, pIVPA/ΔFBR; Gln₁₁₇, pIVPA/Δ FBR/Δ EGF; Gln₁₁₇ ; pIVPA/Δ EGF, Gln₁₁₇ shouldcontain the mutagenized cDNAs encoding compounds 2-1/N-21/Arg,2-1/N-22/Arg and 2-1/N-23/Arg, respectively, now operatively linked tothe polyhedrin promoter. The nucleotide sequence of each mutagenizedcDNA insert may be confirmed by supercoil sequencing with plasmid assubstrate. See e.g., E. Y. Chen et al., 1985, DNA 4(2):165-170.

B. Introduction of the Mutagenized cDNA into the Insect Virus

Each of the pIVPA plasmids containing the mutagenized cDNAs may beintroduced into the insect virus by co-transfection with wild-type AcNPVin Spodoptera cells. 1 ug of purified Autographa californica NPV DNA and10 ug of the desired pIVPA DNA are introduced into Spodoptera cellsgrowing on tissue culture dishes by a calcium phosphate transfectionprocedure (Potter, K. N. and Miller, L. K., J. Invertebr. Path. (1980),36 431-432). The joint introduction of these DNAs into the cells resultsin a double recombination event between the pIVPA plasmid (containingthe mutagenized cDNAs) and the viral DNA at the regions of homologybetween the two; that is, the polyhedrin gene region of the progenyvirus from the recombination event contains the mutagenized cDNA insertfrom the pIVPA plasmid.

Isolation of Virus Containing the Nucleotide Sequence Encoding theProteins of this Invention

The progeny virus present in the media over the transfected cells areplaqued onto a fresh monolayer of cells at several different dilutions.Plaques are assayed, and the recombinants are picked based on thePIB-minus phenotype as follows: A virus which has lost its polyhedringene, as would a virus containing a mutagenized cDNA will not producePIBs. Plaques that appear PIB deficient are selected, excised andamplified on fresh cells. The supernatant over these cells is thenassayed for t-PA-type enzymatic activity. Positive assay resultsindicate that the glycoprotein is in fact being produced.

An alternative method of virus purification via the plaque liftingprotocol differs slightly from the steps described above, and isdescribed below. Plaque the progeny virus from transfection at suitabledilution onto cell culture dishes. Prepare a nitrocellulose replica ofthe cell monolayer and the virus plaques. Reserve the agarose overlayfrom the plate as the virus source after the results of the followingsteps are obtained.

Probe the nitrocellulose filter with radioactive DNA fragmentsrepresentative of the gene being placed into the viral chromosome. Scorepositives as those containing the foreign gene. Remove the hybridizedprobe. Re-probe the filter with radioactive DNA representative of aportion of the viral chromosome removed by substitution with the foreignDNA. One would score positives as those which still have a polyhedringene.

Remove the hybridized probe. Re-probe the filter with a radioactive DNAfragment which will identify viral plaques regardless of the state ofthe polyhedrin gene. A suitable fragment may be the EcoRI I fragment.Score these as progeny virus. Select those plaques which are positivefor the foreign gene DNA probe, negative for the polyhedrin gene probe,and positive for the viral DNA probe. These are strong candidates forthe desired genotype.

C. Production and Characterization of High Mannose Glyco-protein

Antibodies have been used to demonstrate the presence of the variantproteins in the extracellular media of infected cells. Recombinantvirus, prepared as above, is used to infect cells grown in the usualTC-100 (Gibco) nutrient salts solution but instead of the standard mediasupplement of 10% fetal calf serum, this is replaced with a 50% egg yolkenrichment (to 1% total volume) (Scott Biologicals). Previousexperiments had demonstrated a more intact protein under theseconditions. The supernatant from the infected cells is fractionated onan affinity column bearing an attached monoclonal antibody to naturalhuman t-PA. Protein specifically retained by the column is eluted andassayed for t-PA enzymatic activity. A fixed amount of activity units ofthis and control t-PA preparations are separated on an acrylamide gel.This gel is then stained with a silver-based reagent to display theprotein pattern. This reveals that the virus, upon infection of insectcells, leads to the extracellular production of a protein having t-PAtype activity.

Radiolabeled protein is produced for further characterization by firstincubating spodoptera frugiperda cells infected with the virus (m.o.i=1)for 48 hours. The culture plates are then rinsed withmethionine-deficient media. Methionine-deficient media supplemented with³⁵ S-methione is then added to the culture plates. The cell cultures areincubated for 4 hours. The supernatant containing the radiolabeledglycoprotein may be analyzed by SDS-PAGE (7.5%) against wild type (i.e.full-length fully glycosylated) t-PA analogously produced in insectcells and against mammalian t-PA produced e.g. by the method of R.Kaufman et al., Mol. Cell. Biol. 5(7):1750(1985)., but in the presenceof tunicamycin (non-glycosylated). The partially glycosylated truncatedproteins produced in Example 1 should have an increased gel mobilityrelative to the fully-glycosylated analog and to the non-glycosylatedfull-length analog.

EXAMPLE 2 PREPARATION OF OTHER PROTEINS OF THIS INVENTION. A.Preparation of other cDNA's

The mutagenesis methods of Example 1 can be used with otherconventionally prepared synthetic oligonucleotides which modify theoriginal t-PA DNA sequence to produce proteins modified at theN-terminal region and/or optionally modified at N-linked glycosylationsites and/or at Arg-275 with the appropriate codon change(s) describedpreviously. See, e.g.,"Preparation of Mutagenized cDNAs: M13 Method" andRoutes (a)-(h), supra.

For example, cDNA encoding Compounds D-6, D-1 and D-3 may be preparedusing the SacI restriction fragment in M13/t-PA and mutagenizing witholigonucleotides #8, 10 and 12 respectively, but not witholigonucleotide #3. Arg-275 may be deleted or replaced, e.g. with Thr,using oligo's 14 or 15, respectively. Vector construction, transfectionand expression may be carried out as in Example 1 for insect cells or asdescribed below in Example 3 for mammalian cells.

Single-stranded DNA generated from the M13 mutagenesis vector (RFM13/t-PA), prepared as in Example 1, can also be used as a template tomutagenize, in a site specific manner, at Arg-275 and/or atglycosylation site(s) R¹ or R² or both. The region encoding theconsensus tripeptide which encompasses Asn₂₁₈ may be similarlymutagenized. To prepare multiple modifications of the protein at thesesites an iterative process may be used. For example, following theidentification and the purification of M13 phage containing a modifiedR¹ site, single-stranded DNA containing this modified site can bepurified from phage and used as template to initiate a second round ofmutagenesis within the R² site and/or at Arg-275. This process can berepeated until all desired modifications are obtained. Thus, cDNAencoding Compounds 2-2/N-23/Arg, 2-2/N-21/Arg and 2-2/N-22/Arg may beprepared by the method of Example 1 but substituting mutagenesisoligonucleotide #5 for oligonucleotide #3 and screening oligonucleotide#6 for oligonucleotide # 4. cDNA encoding Compounds 2-6/N-21/Arg,2-6/N-22/Arg and 2-6/N-23/Arg may be prepared by twice mutagenizing theSacI fragment as described in Example 1 and addition mutagenizing andscreening with oligonucleotides #5 and #6. Vector construction,transfection and expression are carried out as in Example 1 for insectcells or as described below for mammalian cells. See Routes (a)-(h),supra.

The RF M13/t-PA mutagenesis vector does not contain DNA sequenceencoding R³, the N-linked glycosylation site of t-PA most proximal tothe carboxy-terminus of the protein. Therefore in order to make DNAmodifications at that site, a new M13/t-PA mutagenesis RF vector calledM13/t-PA:R1-Xma I was made. This vector was constructed by digesting theM13 vector M13mp11 to completion with EcoRI and Xma I. The R1/XmaIdigested M13 vector was ligated to a purified EcoRI/Xma I t-PArestriction fragment (approximately 439 bp, hereinafter 0.4 kbp)encoding a polypeptide region encompassing glycosylation site R³. This0.4 kbp restriction fragment was purified following digestion of theplasmid pWGSM with EcoRI and Xma I. The mammalian expression plasmidpWGSM, encoding the t-PA gene, is identical within the 439 bp EcoR1/XmaI fragment to the plasmid pLDSG described by Kaufman et al., Mol. CellBiol. 5:1750-1759 (1985).

The ligation mixture was used to transform competent bacterial JM 101cells. Several plaques were picked and analyzed for the presence of the0.4 kpb t-PA EcoRI/XmaI fragment by standard DNA restriction fragmentanalysis. Double-stranded RF M13 DNA was purified from cells containingthe 0.4 kbp t-PA fragment. This DNA was designated RF M13/t-PA:RI-Xma Imutagenesis vector. As previously indicated in Example 1A this vector,when transformed into competent JM101 cells, can be used to makeM13/t-PA:RI-XmaI phage from which single-stranded M13/t-PA:RI-XmaI DNAcan be purified. This single-stranded DNA can be used as template in thesite-directed mutagenesis reaction to modify the t-PA DNA at theN-linked glycosylation site R³.

Modified R³ coding sequences can be used to replace the wild-type R³sequences present in either modified pIVPA/1 as prepared in Example 1(truncated and/or modified at R¹ and/or R²) or wild-type pIVPA/1 plasmidDNA. This can be accomplished by first performing a total Sac I/Apa Idigestion of the R³ modified M13/t-PA:RI/XmaI mutagenesis plasmidvector, and isolating the R³ modified 165 base pair t-PA restrictionfragment so produced. The insect expression vector pIVPA/1 or pIVPA/1plasmid DNA modified, e.g. as in Example 1, can similarly be totallydigested with Sac I and Apa I to excise the 165 bp wild-type t-PArestriction fragment encoding the unmodified R³ site. Ligation of thepurified insect expression vector lacking the 165 bp fragment to themodified R³ 165 bp fragment produces a new insect expression vector.Expression of the vector produces a truncated protein modified at the R³site, as well as at any or all of the other consensus N-linkedglycosylation sites present in natural t-PA and/or at Arg-275.

The pIVPA plasmid containing the modified cDNA may also be used togenerate the BglII/ApaI fragment of the modified t-PA cDNA which spansthe deletion region in the N-terminal domain as well as the regionencoding R¹, R² and R³ or the NarI/XmaI fragment which spans R¹, R² andR³. Either of those fragments may be inserted into mammalian expressionvectors such as pSVPA4 or pWGSM as described in Example 3.

EXAMPLE 3 PREPARATION OF COMPOUNDS D-6, D-1 and D-3 in MAMMALIAN CELLSA. Preparation of cDNA.

cDNA molecules encoding the polypeptide sequences of compounds D-6, D-1and D-3 were prepared using mutagenesis oligonucleotides #8, 10, and 12,respectively, and the SacI fragment of the t-PA cDNA as template by theM13 method of Example 1 or heteroduplex mutagenesis (MoranagaHeteroduplex Mutagenesis protocol; both, supra). Mutants selected by DNAhybridization using screening oligonucleotides 9, 11 and 13 respectivelywere confirmed by DNA sequence analysis to be correct in the modifiedDNA sequence.

B. Modified t-PA Vector Preparation

Each modified cDNA prepared in Example 1A (Δ, Gln₁₁₇) or 3A (Δ) wasfirst removed from the M13 mutagenesis vector RF M13/t-PA by totaldigestion of the vector with SacI. The approximately 1.4 kbp restrictionfragment of each mutagenized cDNA was purified by gel electrophoresisand then ligated into pSVPA4 as follows. First, pSVPA4 was digested withSacI to remove the wild type t-PA 1.4 kbp restriction fragment. Theremaining portion of the SacI digested pSVPA4 was then ligated to the1.4 kbp restriction fragment of the mutagenized cDNA. This ligationevent can produce two orientations of the inserted fragment. Theappropriate orientation in each case may be identified using EcoRI andPvuII as the enzymes in conventional analytical restriction enzymeanalysis. This replacement allows the Sac I fragment to be used as acassette fragment between the RF M13/t-PA mutagenesis vector and thepSVPA4 mammalian expression vector. Modified M13 SacI fragments(truncated and optionally modified at R¹ and/or R²) may be inserted intoSacI-digested pSVPA4 DNA which has been previously, or is subsequently,modified at R³ if desired. Alternatively, DNA previously modified at R¹,R² and/or R³ can be excised from vectors such as pIVPA or pSVPA4 as aNarI/ApaI or NarI/XmaI fragment. The fragment so obtained may then beinserted into vectors such as pSVPA4 or pWGSM previously digested withNarI (partial) and ApaI or XmaI (total). By this method any combinationof N-terminal deletion and/or substitution and/or glycosylation sitemutagenesis and/or Arg-275 mutagenesis may be achieved.

C. Transfection of COS (SV40 transformed African Green Monkey Kidney)Cells

COS-1 cells (ATCC CRL 1650) were transfected by the method of Lopata, M.A. et al., Nucl. Acids Res. 12:5707-5717 (1984) with the vectorsprepared in Example 3B, i.e., modified pSVPA4. Serum containing mediumwas replaced with serum-free medium 24 hours after the transfection andconditioned medium was assayed for both the presence of plasminogenactivating activity, using the chromogenic substrate S-2251, or thepresence of t-PA antigen by an ELISA assay, 48 and 72 hourspost-transfection.

D. Viral propagation in CV1 (African Green Monkey Kidney) cells.

Modified complex carbohydrate protein can be produced by infecting CV1cells (ATCC CCL 70) with SV40 viral stocks propagated as described byGething and Sambrook (Nature 293:620-625, 1981). This has been carriedout by first totally digesting modified pSVPA4 with the restrictionendonuclease BamH1 to remove the bacterial shuttle vector pXf3 from theSV40 viral DNA. Before transfecting this DNA into CV1 cells, along withthe helper virus SV40-rINS-pBR322 DNA (described below), the Bam HIlinearized SV40/t-PA DNA is circularized by ligation at dilute DNAconcentrations (1 ug/ml). This process was repeated with the insulincontaining SV40 vector SV40-rINS-pBR322 (Horowitz, M. et al., 1982,Eukaryotic Viral Vectors, pp. 47-53, Cold Spring Harbor Laboratory). Thebacterial shuttle vector pBR322 in SV40-rINS-pBR322 was removed by atotal EcoRI digestion. The linearized insulin/SV40 viral DNA was thencircularized by ligation at a DNA concentration of 1 ug/ml. It isnecessary to transfect CV-1 cells with circular ligated pSVPA4 andSV40-rINS DNAs, at equimolar amounts in order to generate viral stocks.SV40-rINS is used to provide"late" SV40 proteins while pSVPA4 providesthe"early" SV40 proteins necessary for virus DNA production while alsoencoding the proteins of this invention. Consequently when cells aretransfected with both these DNA's as described by Gething and Sambrook,SV40 virus is produced which contains DNA from either viral vectors.Subsequent infection of CV1 cells with amplified virus has producedprotein with t-PA-type activity which can be assayed 72 hourspost-infection as described in Example 3C.

EXAMPLE 4 Preparation of Other Proteins

cDNAs encoding various proteins of this invention have been prepared bythe methods of Examples 1, 2 and 3. The Bgl II/XMaI restriction fragmentcassette may then be excised from either the pIVPA or pSVPA4 vectorcontaining the cDNA encoding the truncated protein with or withoutmodification at one or more glycosylation sites. The excised BglII/XmaIfragment may then be ligated into Bgl II/XmaI-cut pSVPA4 or pWGSM forintroduction into mammalian cells. Expression of such cDNAs in mammalianhost cells, e.g. by the method of Example 3 or by the method of Kaufmanet al., supra, (CHO host cells) or by the method of Howley et al., U.S.Pat. No. 4,419,446 (1983) (BPV expression systems) yields thecorresponding mammalian-derived truncated proteins. Thus, cDNAs encodingcompounds 2-1/N-21/Arg (ΔFBR, Gln₁₁₇) and 2-1/N-22/Arg (ΔFBR/EGF,Gln₁₁₇) were prepared and inserted into pSVPA4 as described above. cDNAencoding compound 2-1/N-23/Arg (ΔEGF, Gln₁₁₇) was prepared usingmutagenesis oligonucleotide #12 and screening oligonucleotide #13 (Table7) but by the heteroduplex method described above, with pSVPA4previously mutagenized at position 117 (as above) as template.Similarly, cDNAs encoding Compounds D-1 (ΔFBR) and D-3 (ΔEGF/FBR) wereprepared by M13 mutagenesis, as described above, and inserted as theSacI fragment into SacI-digested pSVPA4. cDNA encoding Compound D-6(ΔEGF) was prepared by the heteroduplex method, described above, usingpSVPA4 as template and mutagenesis oligonucleotide #12, and screeningwith oligonucleotide #13.

To prepare the cDNAs encoding the proteins for amplification andexpression in mammalian cells, cDNA contained in pSVPA4 or pIVPA isexcised as a BglII/XmaI fragment and ligated into purified,BglII/XmaI-digested pWGSM. In each case the resulting pWGSM vector isintroduced into CHO cells and amplified by the method of Kaufman, supra.The transformed and amplified CHO cells produce compounds D-6, D-1, D-3,2-1/N-23/Arg, 2-1/N-21/Arg and 2-1/N-22/Arg respectively, which weredetected in the culture medium by human t-PA specific antibodies. Thecompounds may then be recovered and purified by immunoaffinitychromatography.

EXAMPLE 5

Example 4 may be repeated using cDNA encoding the proteins modifiedwithin the N-terminus and/or at R-275 with or without modification atR¹, R², and/or R³ to produce the desired protein in CHO cells.Mutagenized cDNAs may be prepared as described above. Thus, cDNAsencoding Compounds 2-7/N-23/Arg, 2-7/N-21/Arg and 2-7/N-22/Arg areprepared in pIVPA as described in Example 2. The cDNAs may then beexcised as the BglII/XmaI fragment and ligated into purified,BglII/XmaI-digested pWGSM, and the resultant vector transformed andamplified in CHO cells as in Example 4 to produce compounds2-7/N-23/Arg, 2-7/N-21/Arg and 2-7/N-22/Arg.

What is claimed is:
 1. A thrombolytic protein consisting essentially ofthe peptide sequence of human t-PA, wherein:(a) Cys-6 through Ile-86 aredeleted, and (b) the N-linked glycosylation sites at position 117-119,184-186 and 448-450 are modified such that Asn-117, Asn-184 and Asn-448are replaced with Gln.
 2. The thrombolytic protein of claim 1, whereinthe amino acid at position 245 of the peptide sequence is Met or Val.